Manganese Induces Oxidative Impairment in Cultured Rat Astrocytes

TLDR

Excessive free radical formation, implicated in neurotoxic damage from metals such as manganese, is exacerbated by Mn accumulation in astrocytes, impairing their capacity to modulate neuronal function. The study aimed to assess how Mn exposure affects astrocyte oxidative injury, ATP levels, mitochondrial membrane potential, glutamine uptake, and transporter expression. Oxidative damage was quantified by measuring F₂‑isoprostanes using stable‑isotope dilution GC‑MS. Mn exposure rapidly increased F₂‑isoprostanes, decreased ATP and mitochondrial potential, inhibited glutamine uptake and transporter mRNA, demonstrating that oxidative stress, mitochondrial dysfunction, and disrupted glutamine cycling underlie Mn neurotoxicity.

Abstract

Excessive free radical formation has been implicated as a causative factor in neurotoxic damage associated with exposures to a variety of metals, including manganese (Mn). It is well established that Mn accumulates in astrocytes, affecting their ability to indirectly induce and/or exacerbate neuronal dysfunction. The present study examined the effects of Mn treatment on the following endpoints in primary astrocyte cultures: (1) oxidative injury, (2) alterations in high-energy phosphate (adenosine 5'-triphosphate, ATP) levels, (3) mitochondrial inner membrane potential, and (4) glutamine uptake and the expression of glutamine transporters. We quantified astrocyte cerebral oxidative damage by measuring F(2)-isoprostanes (F(2)-IsoPs) using stable isotope dilution methods followed by gas chromatography-mass spectrometry with selective ion monitoring. Our data showed a significant (p < 0.01) elevation in F(2)-IsoPs levels at 2 h following exposure to Mn (100 microM, 500 microM, or 1 mM). Consistent with this observation, Mn induced a concentration-dependent reduction in ATP and the inner mitochondrial membrane potential (DeltaPsi(m)), measured by the high pressure liquid chromatography method and the potentiometric dye, tetramethyl rhodamine ethyl ester, respectively. Moreover, 30 min of pretreatment with Mn (100 microM, 500 microM, or 1 mM) inhibited the net uptake of glutamine (GLN) ((3)H-glutamine) measured at 1 and 5 min. Expression of the messenger RNA coding the GLN transporters, SNAT3/SN1 and SNAT1, was inhibited after 100 and 500 microM Mn treatment for 24 h. Our results demonstrate that induction of oxidative stress, associated mitochondrial dysfunction, and alterations in GLN/glutamate cycling in astrocytes represent key mechanisms by which Mn exerts its neurotoxicity.